Fbxo42 promotes the degradation of Ataxin-2 granules to trigger terminal Xbp1 signaling

Abstract

The Unfolded Protein Response (UPR) is activated by the accumulation of misfolded proteins in the Endoplasmic Reticulum (ER), a condition known as ER stress. Prolonged ER stress and UPR activation cause cell death, by mechanisms that remain poorly understood. Here, we report that regulation of Ataxin-2 by Fbxo42 is a crucial step during UPR-induced cell death. From a genetic screen in Drosophila, we identify loss of function mutations in Fbxo42 that suppress cell death and retinal degeneration induced by the overexpression of Xbp1^spliced, an important mediator of the UPR. We identify the RNA binding protein Ataxin-2 as a substrate of Fbxo42, which, as part of a Skp-A/Cullin-1 complex, promotes the ubiquitylation and degradation of Ataxin-2. Upon ER-stress, the mRNA of Xbp1 is sequestered and stabilized in Ataxin-2 granules, where it remains untranslated. Fbxo42 recruitment to these granules promotes the degradation of Ataxin-2, allowing for the translation of Xbp1 mRNA and triggering cell death during the terminal stages of UPR activation.

Fig. 1. Loss-of-function mutations in Fbxo42 suppress glossy eye phenotype caused by overexpression of Xbp1^spliced.

a Wild-type adult Drosophila eye (Canton S). Posterior is to the right and dorsal to the top in this and all subsequent panels. Scale bar = 200 μm. b Glossy eye phenotype caused by overexpression of Xbp1^spliced (Xbp1s). Genotype: GMR-GAL4, UAS-Xbp1s, UAS-DsRed. c Glossy eye phenotype is suppressed by co-expression of Xbp1^unspliced (Xbp1u). Genotype: GMR-GAL4, UAS-Xbp1s, UAS-Xbp1u. d Glossy eye phenotype is suppressed by co-expression of caspase inhibitor P35. Genotype: GMR-GAL4, UAS-Xbp1s, UAS-P35. e Glossy eye phenotype is suppressed by co-expression of DIAP1. Genotype: GMR-GAL4, UAS-Xbp1s, UAS-DIAP1. f Genetic scheme of the FLP/FRT F1 genetic screen for suppressors of the glossy eye phenotype caused by overexpression of Xbp1s. g Glossy eye phenotype is suppressed by clones of suppressor 209. Genotype: eyFlp,GMR-GAL4,UAS-Xbp1s; FRT42D, Su209/FRT42D, ubiGFP. h Glossy eye phenotype is suppressed by clones of suppressor 218. Genotype: eyFlp,GMR-GAL4,UAS-Xbp1s; FRT42D, Su218/FRT42D, ubiGFP. i Schematic representation of Fbxo42 (amino acids 1–667), with the F-box domain (blue box) and the mutations found in six of the suppressor alleles (Su209, Su212, Su217, Su218, Su226 and Su359) obtained from the genetic screen. Su212, Su217 and Su218 have a small deletion that causes a frameshift and premature Stop codon at 423. j Overexpression of Fbxo42-GFP abolishes suppression of glossy eye by Su209. Genotype: eyFLP,GMR-GAL4,UAS-Xbp1s; FRT42D, Su209/FRT42D, ubiGFP; UAS-Fbxo42-GFP. k A genomic rescue construct containing Fbxo42 (Pacman CH322-12H15) abolishes suppression of glossy eye by Su359. Genotype: eyFLP,GMR-GAL4,UAS-Xbp1s; FRT42D, Su359/FRT42D, ubiGFP; CH322-12H15. l Immunofluorescence of 3^rd instar larva eye discs containing clones of Su218, labeled by the absence of ubiGFP (green). GMR-GAL4 driven expression of UAS-DsRed is similar in Su218 and ubiGFP clones. Genotype: eyFlp,GMR-GAL4, UAS-DsRed; FRT42D, Su218/FRT42D, ubiGFP. Three independent replicates were conducted. Scale bar = 30 μm. m Glossy eye phenotype caused by overexpression of Rh1. Genotype: eyFlp,GMR-GAL4; FRT42D/FRT42D, GMR-hid, CL;UAS-Rh1. n Whole eye mutant clones of Su218 suppress glossy eye phenotype caused by overexpression of Rh1. Genotype: eyFlp,GMR-GAL4; FRT42D, Su218/FRT42D, GMR-hid, CL;UAS-Rh1. o Small eye phenotype caused by overexpression of hid. Genotype: eyFlp,GMR-hid; FRT42D/FRT42D,ubiGFP. p Clones of Su218 do not suppress the small eye phenotype caused by overexpression of hid. Genotype: eyFlp,GMR-hid; FRT42D, Su218/FRT42D,ubiGFP. q Immunofluorescence of 3^rd instar larva eye discs containing clones of Su218, labeled by the absence of ubiGFP (green), with an antibody made against Fbxo42 (Red). DAPI is in blue. Genotype: eyFlp,GMR-GAL4; FRT42D, Su218/FRT42D, ubiGFP. Scale bar = 60 μm. Three independent replicates were conducted.

Fig. 2. Fbxo42 promotes Ataxin-2/Ubiquitin conjugates in Drosophila eyes and S2 cells.

a Volcano plot of proteins identified by mass spectrometry after biotin/streptavidin pulldowns from Drosophila adult heads expressing ^bioUb (ubiquitin with biotinylation acceptor site) and Fbxo42 or Fbxl7, under the control of GMR-GAL4 driver. Results are presented as log₂ LFQ (label free quantitation) intensity ratios. As blue dots are ACC (acetyl-coenzyme A carboxylase) and PCB (pyruvate carboxylase), endogenous proteins which are biotinylated naturally and should have a log₂ ratio of around 0, if similar amount of biological samples have been isolated during the pulldown. Also as blue dots are Ubiquitin, Fbxl7 and Fbxo42. As green and red dots are proteins identified by at least 2 unique peptides, which are either enriched or depleted, respectively. Ataxin-2 (Atx2) is highlighted by a blue dashed circle. Statistical analysis was performed by two-sided Student’s t test. The full results are provided in Supplementary Data 1. b Immunoblot from Drosophila adult heads expressing ^bioUb, Fbxo42 and Ataxin-2-HA, under the control of GMR-GAL4 driver. Drosophila head lysates were subjected to biotin/streptavidin pulldowns and immunoblot with anti-HA. DTT sensitive Ataxin-2-HA ^bioUb conjugates are detected by an upwards shift of the bands on the gel. n = 3 of biologically independent experiments.c Immunoblot of protein extracts from Drosophila S2 cells expressing His-myc-Ub, Ataxin-2-GFP and in the presence or absence of RNAi against Fbxo42. S2 cells lysates were subjected to Histidine (His) pulldown and immunoblots with anti-GFP (top panels) or anti-myc (bottom panels). DTT sensitive Ataxin-2-GFP His-myc-Ub conjugates are detected by an upwards shift of the bands on the gel and are not observed upon Fbxo42 RNAi treatment. * indicates residual His-myc-Ub-independent pulldown of Ataxin-2-GFP, presumably by direct interaction with the nickel resin. n = 2 of biologically independent experiments. Source data for figures (b, c) are provided as Source Data file. d Glossy eye phenotype caused by overexpression of Xbp1^spliced under the control of GMR-GAL4. Genotype: eyFlp,GMR-GAL4,UAS-Xbp1s/FM7. e Glossy eye phenotype is reduced in clones of Su218. Genotype: eyFlp,GMR-GAL4,UAS-Xbp1s; FRT42D, Su218/FRT42D, ubiGFP. f RNAi for Ataxin-2 suppresses the reduction of the “glossy” eye phenotype by clones of Su218. Genotype: eyFlp,GMR-GAL4,UAS-Xbp1s; FRT42D, Su218/FRT42D, ubiGFP; UAS-Ataxin-2 RNAi.

Fig. 3. Fbxo42 interacts with Ataxin-2 in Drosophila tissues and S2 cells.

a Fbxo42 co-immunoprecipitates with Ataxin-2-GFP in S2 cells. Immunoblots probed with anti-Fbxo42 and anti-GFP antibodies from protein extracts of S2 cells expressing Ataxin-2-GFP before/after immunoprecipitation with anti-GFP beads and with/without Fbxo42 RNAi treatment. n = 2 of biologically independent experiments. b Immunofluorescence of ring gland (3^rd instar larva) showing uniform staining of Ataxin-2 (green) and Fbxo42 (red). DAPI (blue) is a marker for nuclei. Scale bar = 30 μm. c Inset of (b). d Immunofluorescence of ring gland (3^rd instar larva) after 4 h treatment with DTT (5 mM), to induce ER stress, shows Ataxin-2 (green) aggregates decorated with Fbxo42 (red). e Inset of (d). White arrows indicate examples of Ataxin-2 (green) aggregates decorated with Fbxo42 (red). f Inset of (e). Ataxin-2 granule (green), indicated with dashed line is decorated with several foci of Fbxo42 (red). g Quantification of the number of granules containing Ataxin-2 only (green bar) or Fbxo42-decorated Ataxin-2 granules (yellow bar) present in untreated ring gland cells (shown in (b)) and in ring gland cells treated with 5 mM DTT for 4 h (shown in (d)). The quantification was done in 2 biological replicates per condition and is presented in percentage (%) as mean ± SD. Two-way ANOVA coupled with Sidak’s multiple-comparison test, ****p < 0.0001. The number of granules scored in untreated ring glands was n = 277 (replicate 1) and n = 269 (replicate 2). The number of granules scored in ring gland cells treated with DTT was n = 552 (replicate 1, from 15 cells) and n = 523 (replicate 2, from 11 cells). Source data for figures (a, g) are provided as Source Data file.

Fig. 4. Fbxo42 promotes the degradation of Ataxin-2.

a Immunoblots probed with anti-Fbxo42 and anti-GFP antibodies from protein extracts of Drosophila S2 cells expressing Ataxin-2-GFP and FLAG-HA-Fbxo42 or Ataxin-2-GFP and empty vector negative control. S2 cells were treated with cycloheximide to inhibit protein translation and protein extracts were “chased” at the indicated time points. Tubulin was used as loading control and it was detected on the same membrane as Ataxin-2-GFP and Fbxo42. b Quantification of Ataxin-2-GFP protein levels from (a) and 2 other biologically independent experiments (n = 3) is presented as mean ± SEM. Two-way ANOVA coupled with Sidak’s multiple-comparison test, **p = 0.0027 at 6 h for Ataxin-2-GFP+empty vector (blue circles) vs Ataxin-2-GFP+FLAG-HA-Fbxo42 (green squares); *p = 0.0322 at 12 h for Ataxin-2-GFP+empty vector vs Ataxin-2-GFP+FLAG-HA-Fbxo42. c Immunoblots probed with anti-Fbxo42 and anti-GFP antibodies from protein extracts of Drosophila S2 cells expressing Ataxin-2-GFP and Fbxo42 RNAi or Ataxin-2-GFP and LacZ RNAi, as negative control. S2 cells were treated with cycloheximide to inhibit protein translation and protein extracts were “chased” at the indicated time points. Tubulin was used as loading control and it was detected on the same membrane as Ataxin-2-GFP. d Quantification of Ataxin-2-GFP protein levels from c and 1 other biologically independent experiment (n = 2) is presented as mean ±  SEM, where Ataxin-2-GFP+Fbxo42 RNAi is represented by green squares and Ataxin-2-GFP+LacZ RNAi is represented by blue circles. e Immunofluorescence of 3^rd instar larva eye discs containing clones of Su218, labelled by the absence of ubiGFP (green). Endogenous Ataxin-2 is in red and DAPI (blue) is a marker for nuclei. Whole brain/eye disc tissues were treated with 5 mM DTT (in PBS) for 4 h, before fixation with formaldehyde. Genotype: eyFlp, GMR-GAL4; FRT42D, Su218/FRT42D, ubiGFP. Scale bar = 10 μm. f Immunoblots probed with anti-GFP from protein extracts of Drosophila S2 cells expressing Ataxin-2-GFP or Ataxin-2^C244A-GFP. S2 cells were treated with cycloheximide to inhibit protein translation and protein extracts were “chased” at the indicated time points. Tubulin was used as loading control and it was detected on the same membrane as wild-type and mutant Ataxin-2-GFP. g Quantification of Ataxin-2-GFP (WT, blue circles) and Ataxin-2^C244A-GFP (green triangles) protein levels from f and 2 other biologically independent experiments (n = 3) is presented as mean ± SEM. Two-way ANOVA coupled with Sidak’s multiple-comparison test, **p = 0.0087 at 6 h for wild-type Ataxin-2-GFP vs Ataxin-2^C244A-GFP. h Alphafold-3 prediction of a complex containing the LSM and LSM-AD domains (N57 to Q270) of Ataxin-2 (oxblood) together with Fbxo42 (green), Skp1 (yellow), Cullin1 (light blue), Rbx1 (pink), E2-ubiquitin conjugating enzyme (Effete, red) and Ubiquitin (Ub, orange). All proteins are represented as spheres except for Ataxin-2 and Ub that are in ribbon view. The right panel is a zoom in the region highlighted by the black dashed rectangle, with a green highlight in Cys 244 of Ataxin-2, the catalytic Cys 85 of the E2 (Effete) and the reactive C-terminus of Ub (Gly 76). Source data for figures (a–d) and (f–h) are provided as Source Data file.

Fig. 5. Ataxin-2 binds Xbp1 mRNA during UPR activation.

a Immunofluorescence and RNA FISH of untreated ring gland cells (3^rd instar larva). Endogenous Ataxin-2 is in red (anti-Ataxin-2 antibody), Xbp1 mRNA in green (Stellaris FISH probes against Xbp1) and the nuclei is in blue (DAPI). Scale bar = 10 μm. b Immunofluorescence and RNA FISH of ring gland cells treated with 5 mM DTT (to induce ER stress and UPR activation) for 4 h. Endogenous Ataxin-2 (red), Xbp1 mRNA (green) and nuclei (blue), as in (a). Arrows indicate Xbp1 mRNA in Ataxin-2 granules. Arrowheads indicate Xbp1 mRNA in the nucleus. c Quantification of the number of granules of Ataxin-2 protein only (red bar), Xbp1 mRNA only (green bar) or both Ataxin-2 protein and Xbp1 mRNA (yellow bar), in untreated ring gland cells (shown in (a)) and in ring gland cells treated with 5 mM DTT for 4 h (shown in (b)). The quantification was done in 2 biological replicates per condition and is presented in percentage (%) as mean ± SD. Two-way ANOVA coupled with Sidak’s multiple-comparison test, ****p < 0.0001. The number of granules scored in untreated ring glands was n = 125 (replicate 1) and n = 150 (replicate 2). The number of granules scored in ring gland cells treated with DTT was n = 123 (replicate 1) and n = 156 (replicate 2). d iCLIP results for Xbp1/Ataxin-2-HA. In green are depicted the peaks (sequencing reads) in Xbp1 from UV-irradiated S2 cells transfected with Ataxin-2-HA. In blue are depicted the peaks in Xbp1 of UV-irradiated cells transfected with Ataxin-2^C244A-HA. The non-UV irradiated controls for Ataxin-2-HA and Ataxin-2^C244A-HA samples are shown by the yellow and orange lines, respectively. To the right is a zoom of the Xbp1 3’UTR containing the AU-rich region.

Fig. 6. Ataxin-2 and Fbxo42 regulate the stability and translation of Xbp1 mRNA during UPR activation.

a Actinomycin D (ActD) chase experiments using S2 cells treated with Ataxin-2 RNAi (orange triangles) or LacZ RNAi (control, blue circles) and transfected with pUASTattb-Xbp1-HA-GFP. The cells were treated with 5 mM DTT (for 4 h), to induce ER stress, and subsequently incubated with 5 µg/ml ActD until the indicated chase time points. Data are presented as mean ± SD of % of gfp mRNA remaining, normalized to rp49 from biologically independent experiments: LacZ RNAi n  = 3 and Ataxin-2 RNAi n = 2. Half-life for each treatment was calculated by one phase regression analysis (R² LacZ RNAi = 0.9459; R² Ataxin-2 RNAi = 0.9560). Statistical significance was determined for each point: 60 min, p** = 0.026; 120 min, p* = 0.047; 180 min, p = 0.057. b Immunoblot of Xbp1^spliced-GFP (probed with anti-GFP), Xbp1^unspliced-HA (probed with anti-HA) and Ataxin-2 (probed with anti-Ataxin-2) after 5 mM DTT incubation for 0, 4 and 8 h. Tubulin was used as loading control and it was detected on the same membrane as XBP1 and Ataxin-2-HA. S2 cells were treated with LacZ RNAi or Ataxin-2 RNAi and transfected with Xbp1-HA-GFP. c Quantification of Xbp1^spliced-GFP protein levels from (b) and 2 other biologically independent experiments (n = 3) is presented as mean ± SD. Two-way ANOVA coupled with Sidak’s multiple-comparison test, *p = 0.0496 at 4 h for S2 cells treated with LacZ RNAi and expressing Xbp1^spliced-GFP (blue bar) vs S2 cells treated with Ataxin-2 RNAi and expressing Xbp1^spliced-GFP (orange bar); *p = 0.0150 at 8 h for S2 cells treated with LacZ RNAi and expressing Xbp1^spliced-GFP vs S2 cells treated with Ataxin-2 RNAi and expressing Xbp1^spliced-GFP. d Immunoblot of Xbp1^spliced-GFP (probed with anti-GFP), Xbp1^unspliced-HA (probed with anti-HA) and Fbxo42 (probed with anti-Fbxo42) after 5 mM DTT incubation for 0, 4 and 8 h. Tubulin was used as loading control and it was detected on the same membrane as XBP1 and Fbxo42. S2 cells were treated with LacZ RNAi or Fbxo42 RNAi and transfected with Xbp1-HA-GFP. e Quantification of Xbp1^spliced-GFP protein levels from (d) and 1 other independent experiment is presented as mean ± SD. f Immunofluorescence of 3^rd instar larva eye discs containing clones of Su218, labelled by the absence of ubiRFP (red), Xbp1s-GFP is in green and the photoreceptor marker ELAV is in blue. The larval discs were treated with 5 mM DTT for 4 h before fixation to activate Ire1-mediated splicing of Xbp1-HA-GFP. Scale bar = 10 μm. Genotype: eyFlp,GMR-GAL4; FRT42D,ubiRFP/ FRT42D,Su218; UAS-Xbp1-HA-GFP.

Fig. 7. Model of Xbp1 mRNA regulation by Ataxin-2 and Fbxo42.

Upon ER stress, Xbp1 mRNA is transcribed and progressively accumulates in Ataxin-2 (Atx-2) granules, where it is protected from degradation. Ataxin-2 binds Xbp1 mRNA, together with Poly(A)-binding protein (PABP). Fbxo42 binds to Ataxin-2, promoting the ubiquitylation (Ub) of Ataxin-2 and its degradation by the proteosome. Xbp1 mRNA is released from Ataxin-2 granules and is translated by the ribosome.